重金屬鉛單克隆抗體的制備及ELISA檢測(cè)方法的建立
發(fā)布時(shí)間：2019-06-09 17:19
【摘要】： 鉛對(duì)人和動(dòng)物健康的潛在危害,已日益引起人們的關(guān)注。鉛是環(huán)境污染物中毒性很大并且以神經(jīng)毒性為主的一種重金屬元素,其可通過消化道和呼吸道進(jìn)入人和動(dòng)物體內(nèi),對(duì)神經(jīng)、造血、消化、心血管等系統(tǒng)和腎臟造成損害。環(huán)境鉛污染和食品、飼料鉛污染是造成人和動(dòng)物鉛損傷的主要途徑,因此,監(jiān)測(cè)環(huán)境,食品、飼料原料和食品、飼料包裝材料及食品、飼料中鉛的含量,是控制人和動(dòng)物鉛攝入量,預(yù)防和減少鉛對(duì)人和動(dòng)物危害的重要措施。 重金屬離子的免疫學(xué)檢測(cè)方法是一種新型、高效、快速的檢測(cè)方法。與傳統(tǒng)檢測(cè)方法相比該方法具有檢測(cè)速度快、費(fèi)用低廉、簡(jiǎn)單易攜、靈敏度高和選擇性的特點(diǎn)。且可以用于重金屬離子的快速檢測(cè)和常規(guī)檢測(cè),在臨床醫(yī)學(xué)、動(dòng)物檢疫、食品飼料科學(xué)、藥物殘留等領(lǐng)域都有著很廣泛的應(yīng)用前景。 本研究采用重金屬鉛離子與雙功能螯合劑ρ-SCN-CHX-A"-DTPA進(jìn)行螯合反應(yīng),制得Pb2+-SCN-CHX-A"-DTPA螯合物,再將Pb2+-SCN-CHX-A"-DTPA螯合物分別與載體蛋白KLH、BSA偶聯(lián),制備免疫抗原和針對(duì)Pb2+單克隆抗體的檢測(cè)抗原。再使用雙功能螯合劑p-SCN-CHX-A"-DTPA與載體蛋白BSA偶聯(lián),制備檢測(cè)螯合劑的檢測(cè)抗原。 選取6～8周齡BALB/c雌性小鼠4只,腹腔注射合成的免疫抗原Pb2+-SCN-CHX-A"-DTPA-KLH,100μg/只,誘導(dǎo)小鼠機(jī)體產(chǎn)生對(duì)免疫抗原的免疫應(yīng)答,檢測(cè)其所分泌的抗體。 取免疫小鼠脾細(xì)胞與骨髓瘤細(xì)胞(Sp2/0)進(jìn)行融合、篩選及克隆,獲得分泌抗Pb2+單抗的雜交瘤細(xì)胞株,并對(duì)細(xì)胞株所分泌的抗Pb2+單抗的亞類、細(xì)胞株誘生的腹水單抗的濃度及單抗親和常數(shù)進(jìn)行測(cè)定。 在對(duì)單抗的效價(jià)、親和力等進(jìn)行分析的基礎(chǔ)上,選擇單抗2A3用于建立間接競(jìng)爭(zhēng)ELISA檢測(cè)方法,確定反應(yīng)系統(tǒng)最佳工作條件,并建立標(biāo)準(zhǔn)檢測(cè)曲線。 結(jié)果： (1)成功制備了的免疫抗原pb2+-SCN-CHX-A"-DTPA-KLH和檢測(cè)抗原pb2+-SCN-CHX-A"-DTPA-BSA以及針對(duì)螯合劑的檢測(cè)抗原SCN-CHX-A"-DTPA-B SA。(2)檢測(cè)抗原作1：400稀釋(即檢測(cè)抗原濃度為2.5μg/mL)時(shí)反應(yīng)效果最佳,免疫小鼠血清效價(jià)均達(dá)1：16000以上。結(jié)果表明合成的免疫抗原Pb2+-SCN-CHX-A"-DTPA-KLH成功誘導(dǎo)小鼠機(jī)體產(chǎn)生了對(duì)免疫抗原的免疫應(yīng)答,并分泌針對(duì)免疫抗原的特異性抗體。 (3)免疫小鼠脾細(xì)胞與骨髓瘤細(xì)胞(Sp2/0)進(jìn)行融合、篩選及克隆,共得四株抗Pb2+雜交瘤細(xì)胞株,分別命名為IH4、1H7、2H1、2A3。四株抗Pb2+雜交瘤細(xì)胞所分泌的抗體均為IgM亞類。四株雜交瘤細(xì)胞所誘生的小鼠腹水抗體濃度分別為3.85mg/mL,4.21mg/mL,3.98mg/mL,4.08mg/mL,單抗親和常數(shù)2A31H72H11H4,且均在108mol/L以上,單抗具有較高的親和力。由于2A3單抗親和常數(shù)最大,因此抗Pb2+的雜交瘤細(xì)胞株2A3最佳。 (4)建立的間接競(jìng)爭(zhēng)ELISA最佳工作條件為：螯合劑DTPA的最佳工作濃度為4 mM。抗原Pb2+-SCN-CHX-A"-DTPA-BSA (1mg/mL)最佳工作濃度為0.25mg/mL;亢鉛單抗最佳工作濃度為1.02μg/mL;最佳封閉條件為10%兔血清37℃封閉1.5h；二抗最佳工作條件為5000倍稀釋(0.16μg/mL),37℃作用1h；底物最佳作用時(shí)間為15min。在間接競(jìng)爭(zhēng)ELISA最佳工作條件下,建立了鉛離子標(biāo)準(zhǔn)檢測(cè)曲線,其回歸方程為Y=0.4695X+0.0391,相關(guān)系數(shù)R2=0.9878,鉛離子最低檢出濃度為2.072ng/mL。
[Abstract]:The potential harm of lead to human and animal health has attracted more and more attention. Lead is a heavy metal element which is toxic in environmental pollutants and is mainly neurotoxic, which can enter human and animal body through digestive tract and respiratory tract, and can cause damage to nervous, hemopoietic, digestive, cardiovascular system and kidney. lead pollution in the environment, food, feed and lead pollution is the main way to lead to human and animal lead damage, therefore, the content of lead in the monitoring environment, the food, the feed raw materials and the food, the feed packaging material and the food and the feed is the lead intake of the control person and the animal, Important measures to prevent and reduce lead to human and animal hazards. The immunological detection of heavy metal ions is a new, efficient and rapid test. Compared with the traditional detection method, the method has the advantages of high detection speed, low cost, simple and easy carrying, high sensitivity and selectivity, and can be used for rapid detection and routine detection of heavy metal ions, and has wide application in the fields of clinical medicine, animal quarantine, food feed science, drug residue and the like The preparation method of Pb2 +-SCN-CHX-A-DTPA conjugate was carried out by using the heavy metal lead ion and the double-functional iron-binding agent to prepare the Pb2 +-SCN-CHX-A "-DTPA conjugate. The Pb2 +-SCN-CHX-A-DTPA conjugate was then coupled with the carrier protein KLH and BSA respectively to prepare the immune antigen and the detection antigen for the Pb2 + monoclonal antibody. The immune antigen Pb2 +-SCN-CHX-A "-DTPA-KLH,100. m A. the antibody secreted by the cell strain is detected, and the spleen cells of the immunized mouse are fused, screened and cloned in the myeloma cell (Sp2/0) to obtain the hybridoma cell strain which secretes the anti-Pb2 + monoclonal antibody, and the subclass of the anti-Pb2 + monoclonal antibody secreted by the cell line and the ascites sheet induced by the cell strain The concentration of the antibody and the affinity constant of the monoclonal antibody were determined. On the basis of the analysis of the titer and the affinity of the monoclonal antibody, the monoclonal antibody 2A3 was selected to establish the indirect competitive ELISA detection method and the reverse antibody was determined. The system shall be Results: (1) The successfully prepared immune antigen pb2 +-SCN-CHX-A "-DTPA-KLH and test antigen pb2 +-SCN-CHX-A"-DTPA-BSA and the detection antigen SCN-CHX-A "-DTPA-B SA. (2) When the antigen is diluted 1:400 (i.e., the concentration of the test antigen is 2.5. mu.g/ mL), the effect of the reaction is the best, and the serum titer of the immunized mice is 1:16000 or more. The result shows that the synthesized immune antigen Pb2 +-SCN-CHX-A"-DTPA-KLH for the binding agent successfully induced the production of the mouse body (3) The spleen cells of the immunized mice and the myeloma cells (Sp2/0) were fused, screened and cloned to obtain four strains. Pb2 + hybridoma cell strain, named IH4, 1H7, 2H, respectively 1, 2A3. The anti-Pb2 + hybridoma cells of the four hybridoma cells were all IgM subclasses. The concentration of the mouse ascites antibody induced by the four hybridoma cells was 3.85 mg/ mL, 4.21 mg/ mL, 3.98 mg/ mL, 4.08 mg/ mL, and the monoclonal antibody affinity constant of 2 A31H7 2H11H4, and all above 108 mol/ L, and the monoclonal antibody has a high affinity. The affinity constant of the 2A3 monoclonal antibody is the largest, so the anti-Pb2 + hybridoma cell line 2A3 is the best. The optimal working conditions of indirect competitive ELISA were as follows: the optimal working concentration of the mixture of DTPA was 4 mM, the best working concentration of the antigen Pb2 +-SCN-CHX-A "-DTPA-BSA (1 mg/ mL) was 0.25 mg/ mL, the best working concentration of the high-activity lead was 1.02. m The optimum working time of the substrate was 15 min. Under the optimum working condition of indirect competitive ELISA, the standard detection curve of lead ion was established. The regression equation was Y = 0.469X + 0.0391.
【學(xué)位授予單位】：四川農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：S854.4;R392
本文編號(hào)：2495737
[Abstract]:The potential harm of lead to human and animal health has attracted more and more attention. Lead is a heavy metal element which is toxic in environmental pollutants and is mainly neurotoxic, which can enter human and animal body through digestive tract and respiratory tract, and can cause damage to nervous, hemopoietic, digestive, cardiovascular system and kidney. lead pollution in the environment, food, feed and lead pollution is the main way to lead to human and animal lead damage, therefore, the content of lead in the monitoring environment, the food, the feed raw materials and the food, the feed packaging material and the food and the feed is the lead intake of the control person and the animal, Important measures to prevent and reduce lead to human and animal hazards. The immunological detection of heavy metal ions is a new, efficient and rapid test. Compared with the traditional detection method, the method has the advantages of high detection speed, low cost, simple and easy carrying, high sensitivity and selectivity, and can be used for rapid detection and routine detection of heavy metal ions, and has wide application in the fields of clinical medicine, animal quarantine, food feed science, drug residue and the like The preparation method of Pb2 +-SCN-CHX-A-DTPA conjugate was carried out by using the heavy metal lead ion and the double-functional iron-binding agent to prepare the Pb2 +-SCN-CHX-A "-DTPA conjugate. The Pb2 +-SCN-CHX-A-DTPA conjugate was then coupled with the carrier protein KLH and BSA respectively to prepare the immune antigen and the detection antigen for the Pb2 + monoclonal antibody. The immune antigen Pb2 +-SCN-CHX-A "-DTPA-KLH,100. m A. the antibody secreted by the cell strain is detected, and the spleen cells of the immunized mouse are fused, screened and cloned in the myeloma cell (Sp2/0) to obtain the hybridoma cell strain which secretes the anti-Pb2 + monoclonal antibody, and the subclass of the anti-Pb2 + monoclonal antibody secreted by the cell line and the ascites sheet induced by the cell strain The concentration of the antibody and the affinity constant of the monoclonal antibody were determined. On the basis of the analysis of the titer and the affinity of the monoclonal antibody, the monoclonal antibody 2A3 was selected to establish the indirect competitive ELISA detection method and the reverse antibody was determined. The system shall be Results: (1) The successfully prepared immune antigen pb2 +-SCN-CHX-A "-DTPA-KLH and test antigen pb2 +-SCN-CHX-A"-DTPA-BSA and the detection antigen SCN-CHX-A "-DTPA-B SA. (2) When the antigen is diluted 1:400 (i.e., the concentration of the test antigen is 2.5. mu.g/ mL), the effect of the reaction is the best, and the serum titer of the immunized mice is 1:16000 or more. The result shows that the synthesized immune antigen Pb2 +-SCN-CHX-A"-DTPA-KLH for the binding agent successfully induced the production of the mouse body (3) The spleen cells of the immunized mice and the myeloma cells (Sp2/0) were fused, screened and cloned to obtain four strains. Pb2 + hybridoma cell strain, named IH4, 1H7, 2H, respectively 1, 2A3. The anti-Pb2 + hybridoma cells of the four hybridoma cells were all IgM subclasses. The concentration of the mouse ascites antibody induced by the four hybridoma cells was 3.85 mg/ mL, 4.21 mg/ mL, 3.98 mg/ mL, 4.08 mg/ mL, and the monoclonal antibody affinity constant of 2 A31H7 2H11H4, and all above 108 mol/ L, and the monoclonal antibody has a high affinity. The affinity constant of the 2A3 monoclonal antibody is the largest, so the anti-Pb2 + hybridoma cell line 2A3 is the best. The optimal working conditions of indirect competitive ELISA were as follows: the optimal working concentration of the mixture of DTPA was 4 mM, the best working concentration of the antigen Pb2 +-SCN-CHX-A "-DTPA-BSA (1 mg/ mL) was 0.25 mg/ mL, the best working concentration of the high-activity lead was 1.02. m The optimum working time of the substrate was 15 min. Under the optimum working condition of indirect competitive ELISA, the standard detection curve of lead ion was established. The regression equation was Y = 0.469X + 0.0391.
【學(xué)位授予單位】：四川農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：S854.4;R392
【引證文獻(xiàn)】
相關(guān)碩士學(xué)位論文 前2條
1 吳兵;斑點(diǎn)叉尾洶病毒單克隆抗體及血清免疫球蛋白多克隆抗體的制備及初步應(yīng)用[D];華中農(nóng)業(yè)大學(xué);2011年
2 馬燕玲;抗ω-芋螺毒素MⅦA單克隆抗體的制備與鑒定[D];福建農(nóng)林大學(xué);2013年
，本文編號(hào)：2495737
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